The mechanism of nephrotoxicity and hepatotoxicity of phenacetin are unknown. When phenacetin is incubated with microsomes a reactive acrylating metabolite is produced. Since the hepatotoxicity of acetaminophen has been shown to be mediated through the formation of a reactive metabolite, the production of this metabolite of phenacetin was investigated and compared to the formation of the reactive metabolite of acetaminophen. Both metabolites are produced by a cytochrome P-450 mixed function oxidase. The production of the phenacetin metabolite is stimulated by pretreatment of animals with phenobarbital, whereas, the production of the acetaminophen metabolite is stimulated by pretreatment with 3-methylcholanthrene. When the arylating metabolites of acetaminophen and phenacetin were trapped by including the alternate nucleophile glutathione in the microsomal incubations and both conjugates isolated, they were identical: phenacetin had become de-ethylated. When both glutathione conjugates were trapped in the presence of an oxygen-18 atmosphere and examined by mass spectrometry, oxygen-18 was found to be incorporated into the conjugate of phenacetin, but not into the conjugate of acetaminophen. Thus the arylating metabolite of acetaminophen is formed by N-hydroxylation, whereas the arylating metabolite of phenacetin is formed by epoxidation.